/. Embryol. exp. Morph. Vol. 19, 2, pp. 137-43, April 1968
Printed in Great Britain
\ 37
Differentiation of
alkaline phosphatase and glucose-6-phosphate
dehydrogenase in rat yolk-sac
By E.MARSHALL JOHNSON1 & RALPH SPINUZZI1
From the Department of Anatomical Sciences,
University of Florida College of Medicine
INTRODUCTION
During early gestation in the rat, at the time of rapid embryonic differentiation
and prior to the full formation of the chorioallantoic placenta, the function of
'placental' nutrition was attributed to the yolk-sac by Brunschwig (1927). Such
a paraplacental function of the yolk-sac would assume that nutrients pass
through the parietal wall into the yolk-sac cavity and thence into the embryo
via the visceral yolk-sac epithelium and its underlying vitelline vessels. Supporting this concept were the findings of Everett (1935) who demonstrated in 13-day
embryos that toluidine blue was able to pass into the omphalomesenteric vessels
more rapidly than it could reach the umbilical veins via the chorioallantoic
placenta. Furthermore, the visceral entodermal cells appeared to exert some
selectivity in that trypan blue did not pass into the embryo but was localized in
the apical cytoplasm. More recently, Padykula, Deren & Wilson (1966) demonstrated that the rat yolk-sac concentrated both vitamin B12 and vitamin B12 plus
intrinsic factor throughout most of gestation. Also, the rabbit yolk-sac has been
shown by Deren, Padykula & Wilson (1966) to take up several arnino acids.
During late gestation, from day 19 to term, Brambell & Halliday (1956) demonstrated that maternal antibodies pass into the rat fetus via the yolk-sac
entoderm. The relative importance of the yolk-sac as a 'placental' organ late
in gestation, however, is questioned by the facts that ligation of the omphalomesenteric vessels at this period produces no detrimental effect to the embryo,
and ligation of the umbilical vessels results in embryonic death (Noer &
Mossman, 1947).
Both at the light microscopic and the ultrastructural levels Dempsey (1953)
and Wislocki & Dempsey (1955) observed that the yolk-sac has the characteristics of an absorptive membrane which undergoes continual morphological
and biochemical changes during gestation; these data were confirmed by
1
Authors' address: Department of Anatomical Sciences, University of Florida College of
Medicine, Gainesville, Florida 32601, U.S.A.
138
E.M.JOHNSON & R. SPINUZZI
Padykula (1958) and Padykula & Richardson (1963). Consistent with these
observations were the findings of alteration in the electrophoretically mobile
enzyme forms of lactate and malate dehydrogenase, acid phosphatase, and nonspecific esterase throughout gestation (Johnson & Spinuzzi, 1966). Furthermore,
we demonstrated that when the pregnant rat was fed a synthetic diet deficient
in folic acid but containing the potent teratogen 9-methyl pteroylglutamic acid
the enzymic differentiation of the yolk-sac was selectively altered.
The purpose of the present study was to determine the enzymic differentiation
of the visceral yolk-sac with respect to alkaline phosphatase and glucosesphosphate dehydrogenase, both of which are enzymes implicated by Moog &
Wenger (1952) and Karnofsky (1962) as being important in transport phenomena. The study included visceral yolk-sacs -from normal control pregnancies
and from pregnant rats treated with the folic acid antagonist 9-methyl
pteroylglutamic acid in order to determine the extent to which this teratogenic
procedure was able to alter the normal differentiation of these enzymes.
METHODS
To initiate the period of folic acid deficiency, pregnant black-hooded rats
were intubated with 1 mg of 9-methyl pteroylglutamic acid (9-mePGA) on day 10
of gestation. The vitamin deficiency (Johnson, Nelson & Monie, 1963) was
maintained from day 10 to 13 by a diet of purified foodstuffs which contained
10 mg/100 g of 9-mePGA. The deficiency was terminated by feeding a diet
lacking the antagonist but containing a high level of the crystalline vitamin.
Experimental and normal control rats were killed on days 10, 11, 12, 13, 14,
16, 18 and 20 of gestation. The visceral yolk-sac was dissected free from the
embryos and all remnants of the parietal yolk-sac and chorioallantoic placenta
were removed. The visceral yolk-sacs were pooled, homogenized in an equal
volume of triple-distilled water and subjected to zone electrophoresis in either
starch or polyacrylamide gels.
The equipment and procedure for electrophoretic separation in starch
(Solomon, Johnson & Gregg, 1964) and polyacrylamide gels (Johnson &
Spinuzzi, 1966) have been described previously. Alkaline phosphatase was
visualized by standard biochemical means (Johnson, 1965) with sodium anaphthyl acid phosphate. Isozymes of glucose-6-phosphate dehydrogenase were
visualised with 0-1 M glucose-6-phosphate as substrate employed in conjunction
with 0-2 mg/ml. phenazine methosulfate, 10 mg/ml. nicotinamide adenine dinucleotide phosphate (NADP), and 1 mg/ml. nitro-blue tetrazolium in 0-2 M
phosphate buffer at pH 7-4. Since photographs of similarly stained gels have
been published previously (Johnson & Spinuzzi, 1966), semidiagrammatic
representations of the zymograms were prepared for Figs. 1 and 2.
Differentiation in yolk-sac
139
RESULTS
In yolk-sacs from both normal control and PGA-deficient pregnancies, a
maximum of three mobilities of alkaline phosphatase were resolved by electrophoretic separation in starch gel (Fig. 1). In the controls band number 1 was
uniform in zone width and staining intensity throughout gestation, but band
number 2 appeared on day 13, remained a uniform density to day 16, and then
increased in staining activity on days 18 and 20. Zone number 3 was the slowest
moving of the electrophoretic mobilities. It was uniformly present from days
10 to 13, only weakly reactive on days 14 and 16 and undetected thereafter.
Anodal migration ( + 0) from the origin was detected throughout gestation.
X
X
+o
+o
10
varJ s
11
12
13
14
16
Fig. 1. Diagrammatic representation of alkaline phosphatases from visceral yolksac as seen in starch gels at days 10 to 20 of gestation. For each day studied, the
zones of enzymic activity in normal control yolk-sacs are on the left and those from
PGA-deficient tissues are on the right. Var= a zone of enzymic activity not observed
in every analysis, X = a zone of activity not present in the PGA-deficient tissue
though it was present in normal control yolk-sacs of the same gestational age.
The effects of the folic acid deficiency on this group of enzymes were observed
first on day 11. This was only 24 h after commencement of the PGA-deficient
regimen, but the anodal migration at the origin was absent and band number 1
was undetected in over half of the homogenates. There also occurred a precocious
disappearance of band 3 on days 13 and 14 but staining returned to this area
by day 16 and attained a greater intensity on day 18 than was encountered in
any other stage. It should also be noted that by day 18 normal material no
longer showed any staining in this area. Origin staining was absent on day 11
and was above the control levels on both days 16 and 18.
A maximum of four molecular species of different mobilities of glucosesphosphate dehydrogenase in normal control yolk-sacs was detected by electrophoretic separation in polyacrylamide gel. Band 4 appeared to be the major
isozyme throughout gestation (Fig. 2). It reached its maximum staining reaction
during days 12-14; although this zone persisted through term, it was a minor
140
E.M.JOHNSON & R. SPINUZZI
band on day 16 and day 20. Isozymes numbers 1, 2 and 3 all attained their
greatest staining intensity during the third week of gestation. A positive origin
staining ( + 0) was seen throughout gestation at a relatively constant level but
because of the nature of the homogenate employed in these polyacrylamide gels
it was not considered a definite molecular species.
The effects of folic acid deficiency on the glucose-6-phosphate dehydrogenases
were extensive. This teratogenic treatment from days 10 to 12 produced a 24 h
delay in the appearance of isozyme number 1 and an acceleration of its dis-
11
12
Fig. 2. Diagrammatic representation of glucose-6-phosphate dehydrogenases from
visceral yolk-sac as seen in acrylamide gels.
appearance by day 20. In addition, the staining intensity by this molecule was
reduced from the control levels during the third week. A similar reduction was
caused in zones 2 and 3 in the third week. A more consistent reduction in the
PGA-deficient yolk-sac was detected in isozyme number 4 which equalled or
exceeded the intensity of the controls only on days 16 and 20.
DISCUSSION
Previous histochemical observations by Padykula (1958) have demonstrated
a rise in alkaline phosphatase activity between days 12 and 14 of gestation. (The
day of finding sperm in a smear of vaginal contents is considered as day 0 of
gestation. Literature cited has been altered to standardize comparison of
gestational age.) A second increase in activity was detected on day 16 but was
followed by a precipitous decline by day 19. These findings were confirmed by
the present study to the extent that the greatest number of electrophoretic bands
were present on days 13 to 16. The precipitous decline in activity observed by
Padykula, however, would appear to result from the decline of anodal ( + 0)
staining and the absence of band 3 toward term. Alkaline phosphatase activity
was localized in the brush border and apical cytoplasm of the columnar visceral
Differentiation in yolk-sac
141
entoderm, and in agreement with the observations of Moog & Wenger (1952),
one may conclude that the visceral yolk-sac epithelium has at least part of the
enzyme repertory anatomically associated with transport.
The teratogenic insult used in the present experiments appears to affect the
yolk-sac during the critical period of paraplacental function. The delay in
appearance of band 1 may constitute a major deficit in this enzyme repertory at
the period in which the yolk-sac epithelium appears to be the only available site
for transport. The asynchronyous appearance and disappearance of molecular
forms of alkaline phosphatase in response to the folic acid deficiency is in accord
with previously reported observations for a number of other enzymes (Johnson,
1965; Johnson & Spinuzzi, 1966).
In an extensive study of glycogen storage in the rat placenta (Padykula &
Richardson, 1963) the principal period of glycogen accumulation within the
yolk-sac epithelium was found to be between days 14 and 17 of gestation.
Between 17 and 20 days there was a progressive loss of glycogen within the
epithelial cells, although these cells never became devoid of glycogen. The
suggestion that the yolk-sac serves the function of a fetal liver is difficult to
resolve with the fact that glucose-6-phosphatase could not be demonstrated
within these cells. The suggestion of an intrinsic use for this glycogen (Johnson
& Spinuzzi, 1966) is supported by the high activity of lactate dehydrogenase
within the visceral entoderm. The present findings suggest that the phosphogluconate oxidative pathway (hexose monophosphate shunt) may also be an
active pathway within the visceral yolk-sac. The shunting enzyme, glucose6-phosphate dehydrogenase, reaches its over-all peak activity between days 12
and 18. In addition, staining activity by this enzyme decreases markedly on
day 20 which is in agreement with the time of depletion of glycogen from the
visceral yolk-sac. Karnovsky (1962) has presented evidence that the principal
energy source for phagocytosis in neutrophils is via glycolysis and the phosphogluconate oxidative pathway. Therefore, the presence of active glycolytic and
shunt enzymes within the yolk-sac suggests that these enzymes may be responsible for the energy needs of transport across this membrane. The observation
that the major band of glucose-6-phosphate dehydrogenase reaches a high level
of activity quite early in gestation, which is the period of postulated placental
function for the yolk-sac, also supports the latter suggestion. The teratogenic
insult during early gestation when rapid organogenesis is occurring produces an
enzymic pattern completely foreign to the embryo. A situation other than normal
also occurred in late gestation as indicated by the precocious absence of band 1.
Such paraplasia sequence has been reported previously both on a morphological
and biochemical level (Johnson, 1965).
The possibility exists that the foreign enzyme repertory found in the experimental animals may result in the interference of operation of the hexose monophosphate shunt as a source of 5-carbon sugars for nucleotide and nucleoside
synthesis. The fact that a folic acid-deficient diet causes interference with DNA
10
JEEM 19
142
E. M. JOHNSON & R. SPINUZZI
synthesis (Nelson & Asling, 1962) as well as an independent interference with
cytoplasmic RNA synthesis (Johnson, 1964) lends support to this suggestion.
In view of previous work and the present findings, it appears that the over-all
peak activities of alkaline phosphatase and glucose-6-phosphate dehydrogenase
do not occur on days 10 and 11 when the yolk-sac is probably the major pathway for embryonic-maternal exchange. This fact, however, does not disprove
the concept of paraplacental transport by the yolk-sac, but instead raises
questions for future investigation, i.e. which molecular species of an enzyme are
required for transport, in what direction is the transport at the different gestational ages, and what is the nature of the substances transported ?
SUMMARY
1. Electrophoretic and biochemical techniques were applied to homogenates
of the visceral yolk-sac of rat embryos at various gestational ages.
2. Alkaline phosphatase was shown by starch gel electrophoresis of normal
yolk-sacs to undergo specific sequential changes from day 10 to day 20 of
gestation. The isozyme repertory on any given day of gestation was achieved
by deletions from, or additions to, that of the previous day.
3. Glucose-6-phosphate dehydrogenase was shown, by polyacrylamide gel
electrophoresis of normal yolk-sacs, to achieve early in gestation a qualitatively
constant number of enzyme forms. Quantitative changes among the enzyme
forms occurred as gestation proceeded.
4. A teratogenic folic acid deficiency resulted in the delayed appearance of
some enzyme forms as well as the premature disappearance of others in the two
enzyme systems studied. The effects of the treatment were selective in that not
all of the enzyme forms of the treated material were affected.
RESUME
Differ-enciation progressive de la phosphatase alcaline et de la glucose6-phosphate dehydrogenase dans la vesicule ombilicale du rat
1. Des homogenates de la vesicule ombilicale d'embryons de rats, a des stages
varies de la gestation ont ete soumis a des techniques electrophoretiques et
biochimiques.
2. II a ete ainsi demontre par l'electrophorese sur gel d'amidon que dans la
vesicule ombilicale normale, la phosphatase alcaline subit des changements
progressifs du lOe au 20e jour de la gestation. Le repertoire isozymique de
chaque jour de la gestation differait de celui du jour precedent soit par des
deletions soit par des additions.
3. Par l'electrophorese sur gel polyacrylamide de vesicules ombilicales normales, il a ete montre que la glucose-6-phosphate dehydrogenase se diversifie
tot dans la gestation, en un nombre qualitativement constant de formes en-
Differentiation in yolk-sac
143
zymatiques. Des changements quantitatifs de ces formes enzymatiques ont ete
demontres au fur et a mesure de la progression du developpement.
4. Une deficience teratogenique en acide folique a provoque un retard dans
l'apparition de certaines formes enzymatiques ainsi que la disparition prematuree d'autres de ces formes en ce qui concerne les deux systemes enzymatiques
etudies. Les effets de ce traitement ont ete selectifs en ce sens que toutes les
formes enzymatiques n'ont pas ete affectees.
This research was supported by NIH Grants GM 579 and HD 00109.
REFERENCES
F. W. R. & HALLIDAY, R. (1956). The route by which passive immunity is
transmitted from mother to foetus in the rat. Proc. Roy. Soc. B, 145, 170-8.
BRUNSCHWIG, A. E. (1927). Notes on experiments in placental permeability. Anat. Rec. 34,
237^4.
DEMPSEY, E. W. (1953). Electron microscopy of the visceral yolk sac epithelium of the
guinea pig. Am. J. Anat. 93, 331-64.
DEREN, J. J., PADYKULA, H. A. & WILSON, T. H. (1966). Development of structure and
function in the mammalian yolk-sac. Devi. Biol. 13, 370-84.
EVERETT, J. W. (1935). Morphological and physiological studies of the placenta in the albino
rat. / . exp. Zool. 70, 243-82.
JOHNSON, E. M. (1964). Effects of maternal folic acid deficiency on cytologic phenomena in
the rat embryo. Anat. Rec. 149, 49-56.
JOHNSON, E. M. (1965). Electrophoretic analysis of abnormal development. Proc. Soc. exp.
Biol. Med. 118, 9-11.
JOHNSON, E. M., NELSON, M. M. & MONIE, I. W. (1963). Effects of transitory pteroylglutamic acid (PGA) deficiency on embryonic and placental development in the rat.
Anat. Rec. 146, 215-24.
JOHNSON, E. M. & SPINUZZI, R. (1966). Enzymic differentiation of rat yolk-sac placenta as
affected by a teratogenic agent. / . Embryol. exp. Morph. 16, 271-88.
KARNOVSKY, M. L. (1962). Metabolic basis of phagocytic activity. Physiol. Rev. 42, 143-68.
MOOG, F. & WENGER, E. L. (1952). The occurrence of a neutral mucopolysaccharide at sites
of high alkaline phosphatase activity. Am. J. Anat. 90, 339-78.
NELSON, M. M. & ASLING, C. W. (1962). Unpublished data. (Presented before the second
annual meeting of the Teratology Society, 16-17 March, 1962 at the University of Florida
College of Medicine, Gainesville, Florida.)
NOER, H. R. & MOSSMAN, H. W. (1947). Surgical investigation of the function of the inverted
yolk-sac placenta in the rat. Anat. Rec. 98, 31-7.
PADYKULA, H. A. (1958). A histochemical and quantitative study of enzymes of the rat's
placenta. / . Anat. 92, 118-29.
PADYKULA, H. A., DEREN, J. J. & WILSON, T. H. (1966). Development of structure and function in the mammalian yolk-sac. I. Developmental morphology and vitamin B12 uptake of
the rat yolk-sac. Devi. Biol. 13, 311^8.
PADYKULA, H. A. & RICHARDSON, D. (1963). A correlated histochemical and biochemical
study of glycogen storage in the rat placenta. Am. J. Anat. 112, 215-41.
SOLOMON, E. P., JOHNSON, E. M. & GREGG, J. H. (1964). Multiple forms of enzymes in a
cellular slime mold during morphogenesis. Devi. Biol. 9, 314-26.
WISLOCKI, G. B. & DEMPSEY, E. W. (1955). Electron microscopy of the placenta of the rat.
Anat. Rec. 123, 33-63.
BRAMBELL,
(Manuscript received 5 June 1967, revised 18 September 1967)